Oxidative stress caused by various oxygen containing free radicals and reactive species (collectively called "Reactive Oxygen Species" or ROS) has long been attributed to cardiovascular diseases. In human body, major oxidizing species are super oxide, hydrogen peroxide, hydroxyl radical, peroxy nitrite etc. ROS are produced from distinct cellular sources, enzymatic and non-enzymatic; have specific physicochemical properties and often have specific cellular targets. Although early studies in nineteen sixties and seventies highlighted the deleterious effects of these species, later it was established that they also act as physiological modulators of cellular functions and diseases occur only when ROS production is deregulated. One of the major sources of cellular ROS is Nicotinamide adenine dinucleotide phosphate oxidases (Noxes) that are expressed in almost all cell types. Superoxide and hydrogen peroxide generated from them under various conditions act as signal transducers. Due to their immense importance in cellular physiology, various Nox inhibitors are now being developed as therapeutics. Another free radical of importance in cardiovascular system is nitric oxide (a reactive nitrogen species) generated from nitric oxide synthase(s). It plays a critical role in cardiac function and its dysregulated generation along with superoxide leads to the formation of peroxynitrite a highly deleterious agent. Despite overwhelming evidences of association between increased level of ROS and cardiovascular diseases, antioxidant therapies using vitamins and omega 3 fatty acids have largely been unsuccessful till date. Also, there are major discrepancies between studies with laboratory animals and human trials. It thus appears that the biology of ROS is far complex than anticipated before. A comprehensive understanding of the redox biology of diseases is thus needed for developing targeted therapeutics.

The oxidative stress theory of diseases suggests that free radicals induce damage to biomolecules resulting in their malfunction/dysfunctions followed by the onset of diseases. Oxidative stress has been attributed to a large number of degenerative conditions such as cancer, diabetes, arthritis, atherosclerosis, cardiovascular disorders, Alzheimer's and Parkinson's diseases. [1] Despite attributing this common cause, and widespread evidences suggesting a strong association between oxidative stress, treating those conditions by antioxidants has been largely unsuccessful.

Nevertheless, large progress has been made in past 10 years that highlights the importance of free radicals and reactive species in normal cellular functions. An independent discipline named "Redox biology" has thus emerged. [2]

What are Free Radicals and Reactive Species?

A free radical is defined as a molecule that contains an unpaired electron in its atomic orbit. Free radicals are unstable and highly reactive. The most important oxygen-containing free radicals in our body are superoxide anion radical, hydroxyl radical, nitric oxide (NO) radical and peroxynitrite radicals. Hydrogen peroxide (H 2 O 2 ) is also produced in our body in abundance, but it is not a free radical. However, when it comes in contact with cellular iron, it produces superoxide radical, and it is classified as a reactive species. Accordingly, oxygen-containing free radicals and H 2 O 2 are collectively called reactive oxygen species (ROS). As these are highly reactive, they have the potential damaging effects on all important components of our body, such as lipids, DNA, proteins, and carbohydrates. This leads to cell damage and disturbed homeostasis. ROS are both cellular signals and disease triggers. Their relative excess (oxidative stress) or shortage are harmful. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress.

Sources of free radicals

During cellular respiration in the mitochondria, a small percentage of oxygen (~2%) combines with electrons leaking out of electron transport chain, producing superoxide radicals. Part of this superoxide is immediately converted into H 2 O 2 by mitochondrial superoxide dismutase. Under normal condition, both superoxide and H 2 O 2 generated in mitochondria plays physiological roles by reversively modifying certain regulatory proteins. However, under certain pathological conditions like damaged mitochondria, generation of these ROS increases, causing random oxidative damage of mitochondrial proteins and DNA.

Another source is synthesis by various cells like phagocytic cells, neutrophils and macrophage through specialized enzymes like nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) and myeloperoxidases. These cells produce highly reactive species like peroxynitrite, hydroxyl radical and hypochlorous acid to kill invading pathogens.

Denham Harman in the 1950s first elaborated on the deleterious consequences of free radicals in human health, through the proposition of the "Free radical theory of aging." He suggested that oxygen free radicals are produced during normal respiration, causing damage to tissues. Cumulative damage eventually leads to the loss of function that is manifested as aging followed by death. [3] Discovery of superoxide dismutase, a superoxide scavenging enzyme in 1969 strengthened the concept that free radicals are harmful to cells. In 1972, Harman further modified his original theory and proposed the "Mitochondrial theory of aging," suggesting that ROS produced in the mitochondria (the powerhouse of the cell) is the primary cause of damage to lipids, proteins and mitochondrial DNA. This damage leads to mitochondrial dysfunction and a further increase in ROS production, and cellular damage, accelerating the aging process. Subsequent years, with the emergence of tools for detecting free radicals and reactive species in tissues and cells, along with a better understanding of the pathobiology of diseases, the free radical theory of aging was expanded to include a plethora of age-related disorders including cancer, arthritis, atherosclerosis, other cardiovascular diseases (CVDs), Alzheimer's disease, and diabetes.

The Physiological Role of Reactive Oxygen Specie

The bactericidal function of ROS generated from phagocytic Noxs (these are proteins that move electrons across biological membranes) was reported in the seventies, further emphasizing the deleterious effects of ROS and reactive nitrogen species. [4] A paradigm shift occurred when it was observed that nonphagocytic cells also contain Noxs and superoxide produced from them are bonafide signaling molecules. It is now widely accepted that free radicals and reactive species generated by various enzymatic and nonenzymatic sources act as physiological modulators of cellular functions and tissue degeneration and malfunction occurs only when ROS production is deregulated. Therefore, to understand, how ROS can cause diseases, let us first look into how they regulate cell function [Figure 1].

Among various ROS, H 2 O 2 meets all the requirements to perform a signaling function. First, it is a quite stable inside the cells with a half-life of 10 − 5 s; second, it is a milder oxidant than superoxide and hydroxyl radical; and third, it can be restricted inside the cells in the site of production by antioxidant enzymes, so that it can specifically interact with certain proteins. H 2 O 2 can transiently and reversibly oxidize the cysteine thiols (the compound which is readily available in the treatment of paracetamol poisoning and which is a precursor of glutathione an important endogenous antioxidant) of various regulatory proteins and regulate their functions. By such oxidation of selective cysteines, it can regulate protein functions affecting cell proliferation, differentiation, migration, immune cell activation, apoptosis, etc. At the physiological level, it is involved in vascular remodeling and embryonic development, etc., Since H 2 O 2 is a diffusible molecule, it has long been a question, how its activity is selective rather than cell wide. It is now established that once generated, its cellular location is confined by antioxidant enzymes such as thioredoxin and glutathione peroxidase. These enzymes have several variants located in various cellular compartments and in addition to the attenuation of H 2 O 2 generation, they also reverse the oxidative modifications of protein thiols terminating the signals.

Nicotinamide adenine dinucleotide phosphate oxidases are membrane-associated enzymes that use NADPH as an electron donor to reduce oxygen to superoxide and H 2 O 2 . They constitute a major family of ROS producing proteins involved in health and disease. The prototype member Nox 2 was first described in seventies as the source of the phagocyte respiratory burst. Until date, there are five Nox variants (Nox 1-5) and two more closely related enzymes (DUOX1/2) have been reported. While the Nox enzymes (Nox 1-5 produces superoxide), DUOXes are direct producer of H 2 O 2 . Over activation of Nox 1 and 2 has been associated with the development gastrointestinal inflammation induced in hypertension and restenosis after angioplasty. ROS produced by Nox 5 have been associated with atherosclerosis and cancer.

In the heart, redox signaling plays in important role in several physiological as well as pathological processes. Nox are an important source of ROS associated with cardiac redox signaling pathways. Mostly Nox 2 and Nox 4 are expressed in cardiomyocytes, endothelial cells, fibroblasts, and inflammatory cells. Ischemia, pressure overload, increases Nox 4 levels in heart. Interestingly, Nox 4 produces largely H 2 O 2 and enhances eNOS activity, whereas superoxide radicals disrupts NO signaling. This has important physiological consequences: ROS modulate many signaling pathways that are involved in the development of cardiomyocyte hypertrophy by various agonists like Angiotensin II, endothelin-1, α-adrenoceptor agonists and Nox 2 being the probable ROS source. On the other hand, the role of Nox 4 is quite the opposite. Protection against pressure overload-induced left ventricular hypertrophy was seen in mice with Nox 4 overexpression. Prolonged, severe pressure overload causes heart failure, which involves an increase in mitochondrial ROS, protein oxidation, and mitochondrial DNA damage. [5],[6]

Interstitial fibrosis and extracellular matrix remodeling are integral parts of adverse cardiac remodeling leading to heart failure. Increased renin-angiotensin-aldosterone system and inflammatory pathway activation, along with changes in redox balance are involved in cardiac fibrosis caused by increased load or local tissue injury. ROS trigger dormant growth factors which promotes fibrosis and transcription of profibrotic factors.

From the time since its discovery as an endothelial-derived relaxing factor, NO has emerged as a primary signaling molecule regulating almost all critical cellular functions. NO is needed for normal cardiac function and also plays an important protective role in the ischemic heart disease. [8] NO decreases intracellular Ca 2+ , as well as terminates lipidradical reactions caused by oxidative stress. NO interacts with various components of the mitochondrial respiratory chain regulating cell respiration. It also modulates the mitochondrial generation of ROS, thus can have significant effects on cell survival or death. However, when the cellular level of NO generation is increased, or when cellular ROS production is increased or both, NO can also exert cytotoxic effects, mostly through the formation of peroxynitrite, which acts as a potent mediator of cellular damage. Peroxynitrite interacts (nitration reaction) with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms which in turn causes a variety of cellular responses like oxidative injury, cell necrosis or apoptosis, etc., Nitration of important proteins by peroxynitrites has been documented and may be responsible for cardiac dysfunction. One of these proteins is sarcoplasmic reticulum Ca 2+ -ATPase the nitration of which can cause deranged cardiac contractility. K + channels in the coronary vessels are also prone to be affected by peroxynitrite, which causes impaired coronary flow.

Peroxynitrite generation contributes to myocardial and vascular dysfunction during myocardial ischemic-reperfusion injury, chronic heart failure, and various other CVD conditions. Peroxynitrite generation has also been documented in conditions like diabetes mellitus, cancer, and neurodegenerative disorders.

Peroxynitrite triggers cardiomyocyte apoptosis in programmed cell death, a key modulator mainly in the transition from "compensatory" hypertrophy to heart failure as well as endothelial and vascular smooth muscle cells. Moreover, it upregulates the expression of adhesion molecules in endothelial cells and enhances the adhesion of neutrophils to the endothelium. Peroxynitrite also activates MMPs, which contributes to impaired cardiovascular function in most cardiovascular pathologies.

Evidence of Oxidative Stress in Human Diseases

The most studied effect of oxidative stress in human diseases is on atherosclerosis. [9] Oxidative stress is, therefore, a critical feature in atherogenesis. ROS cause direct damage to the vascular wall, while triggering a number of redox-sensitive signaling pathways, which ultimately cause pro-atherogenic changes. Low-density lipoprotein (LDL) plays a crucial role in atherogenesis. LDLs are deposited in the vascular wall, subsequently are subjected to oxidative stress and then taken up by macrophages, which lead to the formation of foam cells - the characteristic feature of atherosclerosis. Oxidative modification of circulating LDLs occurs primarily in the vascular wall rather than in plasma, which has high natural antioxidant defense mechanisms. The accumulation of foam cells ultimately leads to atherogenesis.

Evaluating Oxidative Stress in Human Cardiovascular Disease

Measurement of stable products released by the oxidative stress reaction is a widely used approach. Serum lipid hydroperoxides, plasma malondialdehyde or urine F2-isoprostanes are commonly used and have been used as a prognostic marker in CVD.

Antioxidants in the prevention of cardiovascular diseases

Once the role of oxidative stress in the pathogenesis was fairly established in the pathogenesis of CVDs, attempts were made over the last few decades, to study the effects of antioxidants (antioxidant vitamins and others) in the primary or secondary prevention of CVDs.

Reports of various meta-analyses (a study design used to systematically assess previous research studies to derive conclusions on the effect of treatment or risk factor for disease) of randomized controlled trials (the gold standard for establishing the evidence for therapeutic benefits) show conflicting evidence.

In a recently published meta-analysis, [10] the association between vitamin or antioxidant supplementation and major cardiovascular events, like cardiovascular death, fatal or nonfatal myocardial infarction, angina, sudden cardiac death, fatal or nonfatal stroke, and transient ischemic attack (cerebral ischemia) was investigated. The 50 trials which fulfilled the inclusion and exclusion criteria of the meta-analysis included 294,478 participants with 156,663 in the intervention groups and 137,815 in control groups. The mean age of the participants ranged from 49 to 82. The year of publication of the included trials ranged between 1989 and 2012. Trials, included in the study were conducted in the United State, the United Kingdom, Finland, Norway, Sweden, France, Italy, Canada, Israel, Australia, China, Germany, Switzerland, and the Netherlands and in 20 countries. The range of supplementation and follow-up periods was 6 months to 12 years.

Among the 50 trials, 30 were primary prevention trials (in subjects who had the risk factors for various CVDs, but did not have any event of the disease), and 20 were secondary prevention trials (patients with coronary heart disease, acute myocardial infarction, unstable angina, transient ischemic attack, stroke).

Meta-analysis of all 50 trials reported no reduction of major cardiovascular events with the use of vitamin or antioxidant supplements (relative risk 1.00; 95% confidence interval 0.98-1.02).

According to the authors of this meta-analysis, there is a discrepancy in findings between animal or in vitro laboratory studies and randomized controlled trials with regard to the protection offered by vitamin or antioxidants (natural forms in fruit and vegetables or synthetic forms) from various CVDs. They proposed various theories, like a) preclinical studies (animal studies and in vitro laboratory studies) might not exactly represent the complexities of the biological processes of the human body and b) the timing of their administration of vitamin or antioxidant supplements might determine the beneficial effects, early versus late interventions may have totally opposite effects.

Conclusions

Oxidative stress plays an important role both in health and disease, including CVDs. But it appears to be too complex an event to be targeted therapeutically to have a translational value. This is reflected by the fact that supplementation with antioxidants in patients with CVD and those with risk factors are not found to have promising therapeutic potentials as prophylactic agents. The apparent discrepancy is presently less than clear. Future long term and larger randomized clinical trials may answer these questions, with more supportive evidence from basic research.